Intensive research on the biological functions of nitric oxide (NO) and other reactive nitrogen oxide species (RNS) demands exogenous sources of NO. NO donors fulfill this need as research tools that can release NO (including its pharmacological equivalents and different redox forms) by a specific mechanism, at a specific time and at a specific location. The main objective of our NO research program is to develop novel NO donors and investigate their interactions with proteins. In the next funding cycle, we will focus on the development of two new classes of NO donors based on our recent research discoveries and preliminary results. N-hydroxyguanidines as NO donors NO generation from N-hydroxyarginine (NHA) is kinetically faster and only requires half the amount of oxygen as compared with L-arginine. Therefore, NHA is a more effective NO donor especially under low oxygen tension. Recently, we found that small N-alkyl-N'-hydroxyguanidines are good NOS substrates with comparable activity to NHA. This finding prompted us to search for more NOS substrates by designing and synthesizing three structurally different N-hydroxyguanidines based on our newly resolved nNOS crystal structures bound with N-alkyl-N'-hydroxyguanidines. Mechanistic studies with some proposed compounds will provide insights into the NOS catalyzed oxidation of N-hydroxyguanidines. Moreover, we propose to explore two types of N-hydroxyguanidino compounds that may show isoform-selectivity as NOS substrates. Enzymatic studies and biological testing will be conducted with the synthesized compounds. Beta-Galaetose-RNS Conjugates In order to target and regulate the delivery of RNS inside a cell of interest, our strategy will be to first transform the cell with E. cob lacZ gene, which encodes beta-galatosidase, and then feed the cell with beta-galactose-RNS conjugates so that RNS will be released intracellularly. We propose to design and synthesize four classes of beta-galactose-RNS conjugates in which beta-galactose is linked to three redox forms of nitric oxide (NO radical, NO + and NO), peroxynitrite (ONOO-) and N-hydroxyguanidines. Using many available cell lines transformed with the reporter lacZ gene from gene therapy research, this set of beta-galactose-RNS conjugates will become new research probes for biological research on NO and other RNS. In addition, N-acetylneuraminic acid conjugated RNS will be synthesized as neuraminidase-catalyzed probes to study the interaction of RNS with viruses.